Antibiotic

ABSTRACT

3-(2-NITRO-3-CHLOROPHENYL) - 4 - CHLOROPYRROLE (CALLED PYRROLNITRIN) AND METHOD OF PREPARATION THROUGH CULTIVATION OF PSEUDOMONAS PYRROCINIA, PSEUDOMONAS AERUGINOSA, PSEUDOMONAS MEPHITICA, PSEUDOMONAS OVALIS AND PSEUDOMONAS SHUYLKILLIENSIS FOLLOWED BY SEPARATION FROM THE CULTURE BROTH. THE COMPOUND IS AN ANTIBIOTIC, USEFUL IN THE TREATMENT OF FUNGAL DIESEASES IN WARM BLOODED ANIMALS.

Oct. 17, 1972 I 51 ARlMA ETAL 3,699,121

ANTIBIOTIC Original Filed March 18, 1965 l 5 Sheets-Sheet 1 I 13 I4 I5 I6 2000 I000 I600 Infrared Spectrum of Pyrrolnitrin (in nujoI) Inventors Zeb arimw, Gaiuzo Tamzuw l/zLrosizL Imam/Ya, fflaowww Wow ii a/rw Twizzdw Oct. 17, 1972 KEI ARIMA ETAL 3,699,121

ANTIBIQTIC Original Filed March 18, 1965 3 Sheets-Sheet 2 F I G. 2 1

I 1 l 350 4 n u l l l l l ULTRAVIOLET SPECTRUM OF PYRROLNITRIN 9 I fizz/anions Wei drzlmw, 641112 Tamara, flu'oski 1W fila sazww io use/K1 61 mm 7M B1W+R awrws Oct. 17, 1972 KEI ARIMA EI'AL 3,699,121

ANTIBIOTIC Original Filed March 18, 1965 3- Sheets-Sheet 5 FIG. 3

. PPM Nuclear Magnetic Resonance Specfrum of Pyrrolnifrin (60Mc in CDCla, inter. ref. T. M.S)

\lez' drim, a/fuzoj mura flimski Imam 1%, M JibZwa/Za/ d/fz'o T'ZZKZZdw United States Patent 3,699,121 ANTIBIOTIC Kei Arima, Gakuzo Tamura, Hiroshi Imanaka, Masanobu Kousaka, and Akio Fukuda, Tokyo, Japan, assignors to Fujisawa Pharmaceutical Co., Ltd.

Continuation of application Ser. No. 440,747, Mar. 18, 1965. This application June 28, 1967, Ser. No. 649,745 Claims priority, application Japan, Mar. 25, 1964, 39/ 16,338 Int. Cl. A61k 21/00 U.S. Cl. 260-3263 1 Claim ABSTRACT OF THE DISCLOSURE 3-(2-nitro-3-chlorophenyl) 4 chloropyrrole (called pyrrolnitrin) and methods of preparation through cultivation of Pseudomonas pyrrocinia, Pseudomonas aeruginosa, Pseudomonas mephitica', Pseudomonas ovalis and Pseudomonas shuylkilliemsis followed by separation from the culture broth. The compound is an antibiotic, useful in the treatment of fungal diseases in warm blooded animals.

This application is a continuation of Ser. No. 440,747 filed Mar. 18, 1965, now abandoned.

This invention relates to a new chemical substance having valuable antibiotic properties. More particularly, this invention relates to a new and useful antibiotic called pyrrolnitrin, and to methods for its production.

In the accompanying drawings, FIG. 1 is the curve of an infrared absorption spectrum of pyrrolnitrin, FIG. 2 is the curve of an ultraviolet absorption spectrum of pyrrolnitrin; and FIG. 3 is the curve of a nuclear magnetic resonance spectrum of pyrrolnitrin.

Now it has been found that the new antibiotic substance can be produced by cultivation of a certain strain of Pseudomonas genus. Preferred strains for the microbial production of pyrrolnitrin are referred to, for convenience, as No. 2327, B-16, No. 29, No. 103 and CB- 416, all of which were isolated by us from soil. After mycological observations of these strains, as will be detailed hereinafter, the strains B-16, No. 29, No. 103 and CB-4l6 were found to be Pseudomonas weruginosav, Pseudomonas mephitica, Pseudomonas ovalis and Pseudomonas shuylkilliensis, respectively. No. 2327 strain was identified as a new strain and named as Pseudomonas pyrrocinia. A culture of the living micro organism has been deposited with and is available from the American Type Culture Collection; it has been designated as ATCC 15958.

ice

TABLE 1.-FORM OF CELLS Rods (rounded): 0.5-0.8 x; Rods (rounded): 0.5-0.7 x; Rods (rounded): 0.5-0.8 x; Rods (rounded): 0.6-0.8 x; Rods (rounded): 0.6-0.75 x;

Patented Oct. 17, 1972 1.4-1.6 u. 1.4-2.0 u. Mostly occurring singly-.." Mostly occurring singly. Mostly occurring singly, but

1.2-2.0 u. 1.2-2.0 u. Occurring slngly. Occurring singly, rarelym pairs and in short sometimes in pairs and in chains short chains. Motile with flagella Motile with a single polar Motile with polar flagella.... Motile with polar flagella.-.. Motile with polar flagella.

flagel1um. N0 Sporulation N0 sponilnhnn No spor No sporulation.

N0 spornlntinn m 0000200 00000000000 I I I l l 00000020 -21}.-- 00 2000000 8820 3020 80 003 -0 0 i 0 b 0 -0 0 0 0 a l. l l m @0008 008000 0 0m 00 00 000 00 03000 mm W830 l ........0 000 0 0 5 o .5 0 0 0. 0 .00 x5 5.....1 0 0 00 00000000505 H -0 3 0500000500 "300.5 30 5 0003 000 000.5 002.500 2000 0a 0028 0000000053 580.00 3 A0 E 0 0 08 000 005000 000 w0w0 I 00 0 0282 @0000 2 0 20 50 .0200 0 0 0 2 "002 000 95200 000.000 00000 30500000 0 020 I 0002002 0 7 0.

"0020000 0.5200 00000 20 2 0 00 00 2000 00 0038 20m 0w50m00000020n. wm mo M2 2 00 2 2 m $0 2 STMO 0200 82 020005 0 220 0 0 0: 03 5. 0200 80:00 02000.% 850220 0 22: 0 0 02 05 .0 8220. 02000.& 52002 0208 02000.5 5803 0 0 0 0 00m 0 0 m0 80 00002000 30000 0 220 00 00 03 5 00 00200020 500020005 mE 000000 00 020:5 5805 30002 00V 003m 200220 M00083 0520 5830 003 0003202 $080.5

-05 000 50805 000002000 0802 830220 0200 00 2220000wn0m 0 0m 00 2000 m0 0083 350M $000005 00:02 E a 2 00 2 m wmwwm 6 0 0 0 m 0 3 8 00 can 0 0 b 38 2: MOQ 00000002 52005 000 5823050 0200 02 000000 0 @020 0 0m 3 00302000: 030m 4580.5 0000 02000: 50250.50 5800 00%0 00 2000 h 00 8 03200 0000. 023:0

. 0000.02 0020MB 00mg 000200 0000 2 2 00a 20 1 008w 02822 08 22:00 0000200 500 0 Z #0002000 800250 8 ma 0 028 0305 M0030 4.58000 0000 02.8 0503 w005w 5800 0000 ew 0 0 0.500 m0 0023 00000 0000000 0 2 0 00 0000020 20.20m 0305 000.05 000002 0000090 0 Z 5800m 0000 00 0.500 mm 0025 000.3 308M020 0200 0000 0282 0000.00 0005MB S 05805 000m 0 5 00 00000 m0 .0 2 0&0 05820 205 0 00m 0258 m 0 0.50 0 0 2 mm 2 00 220 fliflp wwwwwww a 3 00 00 5 0 0 20 2 0 hfiw 000202 0500 0023 4 0 3 00 0.500

225 $5000.50 5805 0000 00 00 0020 Ema 02502 $000000 00 00% 5000000 3 83m 20m 5000200000 005 600000 $502000 02000 $0020 00 020M002 Bm200000w 00000020 02 000 w 00080 0200 0002 0 5 00 8000 m0 .500 :00 0003 0 0E 00 E000 8 03 80200 0000 002.502

tion and acid formation, respectively.

NorE.In the second column of the above table, the symbols Af, fB[ and mean growth, gas evolu- TABLE 3 (TABLE 3-4) N0'rE.-C: Closed; 0: openedj-means neutral or alkaline reaction; means weak acid reaction; means strong reaction.

TABLE 3 (TABLE 3-5) ASSIMILATION OF CARBON COMPOUNDS No. 2327 B-l6 No. 29 No. 103 (113-416 Glucose t Gluconlc acid Salicylic acid m-Hydroxybenzolc acid D-Hydrorybenzolc acid G'entlsinlc 'a'cid. g. Anthranllic acid p-Aminobenzoic acid TABLE 3 (TABLE 345 98-1 (.1961), .36, 663 (1962) and 36, 668 (1962), in view of thefollowing characteristics: 7 (1) Green pigment is formed well.

(2) Growth well at 42 C.

(3) 2-ket0 g1uc0nic acid is produced from glucose.

With reference to the Bergeys manual, No. 29 strain is identified as Pseudomonas mephitica.

With reference to the Bergeys manual, No. 103 strain is identified as Pseudomonas ovalis.

With reference to the Bergeys manual, CB-416 strain is identified as Pseudomonas shuylkilliensis.

Suitable microorganisms for the production of pyrrolnitrin widely covers strains of Pseudomonas genus including the above-illustrated Pseudomonas pyrrocinia, Pseudomonas aeruginosa, Pseudomonas mephitica, Pseudomonas ovalis and Pseudomonas shuylkilliensis as well as their mutants and variants. It is to be understood that there are other Pseudomonas strains suitable for the productioncf pyrrolnitr'in than those specified above and such pyrrolnitrin-producing strains can be easily selected from Pseudomonas genus by those skilled in the art with reference to the physical, chemical and physiological prop erties" of pyi'rolnitrin as detailed hereinafter.

'Fer'mentation process for production of pyrrolnitrin can be conveniently carried out by shake or submerged culture in'an ordinary liquid culture. Not only natural oRow'rHoN THE'SELE'CTIVE [MEDIA BY K. KOMAGATAS METHQD/ No. 2321 B-lfi N0. 29 No.103 (in-416 Glucose plus ammoniums'altrumufll t "-F p-Hydroxybenzoate -l v Glucose plus nitrate --:l: Succinate plus nitrate d: +-l:-\ Glutamate (after 7 days at 30 0)....

In view of the cultural characteristics as above and with reference to Bergeys Manual of Determinative Bacteriology (7th ed.), No. 2327 strain is identified as belonging to Pseudomonas genus. However, it is considered as a new strain and named Pseudomonas pyrrocinz'a by taking into consideration the following characteristics:

(1) Neither water-soluble nor water-insoluble pigments are formed, but 2-keto-gluconic acid is produced from gluconic acid.

(2) Utilization of saccharides, particularly disaccharides, is intense, and an acid is produced from lactose.

(3) In B-C-P- milk test, milk is liquefied at a slightly acidic side.

(4) Optimum pH for growth is about 5-6.

No. B-16 strain, which is identifiable as one of Pseudomonas species with reference to the Bergeys manual, is considered as Pseudomonas aeruginosa with further reference to Journal of the Agricultural Chemistry, Japan 35,

media but also synthetic media may be used as the culture medium. Suitable carbon sources include glycerine, glucose, starch, sucrose, etc. and suitable nitrogen sources are peptone water, meat extract, corn steep liquor, soybean meal, urea, ammonium salts, nitrates, etc. Other inorganic salts, e.g. phosphates, sodium chloride, magnesium sulfate, calcium carbonate, etc., may be added, and the salts comprising ferric or ferrous ions (e.g. ferric sulfate, ferrous sulfate) may be used effectively as inorganic salts to obtain the desired substance in high yield. Suitable defoaming agent, e.g. soybean oil, silicone, etc., can be used if necessary. Ingredients of a culture medium can be optionally determined depending on the type of a strain to be cultured. Usually, cultivation can be carried out at about 25 to about 37 C., and at such temperature, the production of desired antibiotic substance will reach maximum within the period of 2 to 5 days.

Pyrrolnitrin, which is accumulated in the fermentation broth, can be isolated, for example, by extracting the desired substance from the cells with, a suitable solvent, re- 7 moving impurities off, and concentrating the resulting solution .in an appropriate manner. Extraction and purification methods generally used in the field of the antibiotic industry may be used, e.g. solvent extraction method,

carbon process, and absorption chromatography method.

After completion of cultivation, for instance the cells are separated from the culture broth by means of a filter or centrifugal separator and then the separated cells are extracted with acetone or the like with agitation. The extract is purified with active carbon and then concentrated under reduced pressure. The concentrate is extracted again with another solvent, e.g. butyl acetate, petroleum benzine, etc. The extract is washed with aqueous alkali or acid, filtered and then concentrated. The crude oily substance is subjected to chromatography using alumina and then the adsorbed substance is eluted with a suitable solvent to obtain a concentrated fraction from which crude crystalline product can be obtained with methanol-cyclohexane or the like mixed solvent. After-recrystallization from a solvent, pure crystalline product is obtained.

The crystalline *product thus obtained is a neutral substance in the form of pale yellow plates or needles. It has a melting point of 125 C. This is a new chemical compound named 3-(2-nitro-3-chlorophenyl)-4-chloro-pyrrole havingpthestructural formula (1) Both oxidation with 'chromic oxide and ozonolysis of the product yield an acid imide with the empirical formula C H Q N Cl and a melting point of 172 C.

Ultraviolet absorption spectrum of the acid imide shows a characteristic absorption A 310.111,u (e* 287()). and

infrared absorption appears at 1790and -1740 cam- (KBr tablet). .1,

(2) Oxidationof the product with a permanganate yields a' carboxylic acid with the empirical formula u and a melting point of 232C. The carboxylic acid has Cl IIIO:

The elementary analysis of this product shows the fol lowing result:

Calcd. for C I-I O N Cl (percent): C, 46.71 H, 2.33; O, 12.45; N, 10.89; Cl, 27.68. Found (percent): C, 45.68; H, 2.42; o, 12 .41;N, 1o.s2;.c1, 2 7.23. frhe molecular weight of the product is 257. The product'shows no depression in melting point when mixed with an authentic sample of 3-(2-nitro-3-chlorophenyl)-4- chloropyrrole) prepared by synthetic procedures. Other physical and chemical properties of the product will ,be.

given below.

' This product is soluble in methanol, ethanol, n-propa nol, n-butanol, pentanol, methyl acetate, ethyl acetate, butyl acetate, pentyl acetate, acetone, ether, chloroform,

1%, agar 1.5%, pH 6.5) is placed asa' slant the following elementary analysis:

Calcd. (percent): C, 41.60; H, 1.98; O, 31.78'; N,jj6.94; Cl, 17.58. Found (percent): C, 41.78; H, 2.25; 0.31.48; N,7.'18;Cl, 17.33. g Ultraviolet absorption of the c'arboxylic acid isk 283 mu (e'=l030) in ethanol. Infrared absorption of this carboxylicacid appears at 1550 and 1370 cm. (KBr Staphylococcus aureus 209 P -2 6.2 Bacillussubtz'lis PCI 219. 12.5 Escherichia coli 250 Pseudomonas aeruginosa J 250 Mycobacterium 'SP 607' if 250 Aspergillus niger 12.5 Penicillium chrysoger zum 0.2

Trichophyton'asteroides Torula utilis Candida albicans ;f: 15:01

In the bioassay of a culture potency, Trichophyto'ri ds teroides and Penicillium chrysogenum are preferredtest microorganisms. Sabougraud agar v(glucose.4%, 'peptone dium in a testitube. After inoculation, 'incub'ati ed at 30 C. for 7 days. 215 ml. of sterilewater 'is' 'added and .cells .are scraped by a platinum..loop as form a cell suspension. A; suitable amount of the cell suspension is added to Sabougraudagar, andstirred uniformly. .Using glacial acetic acid, acetic anhydride, petroleum' ether petroleum benzine and ligroin, and sparingly soluble in water and cyclohexane. With respect to the coloring reactions of the product, it is. positive in ninhydrin reaction (brown) and in both Tollens and Pauly reactions (red), and negative'in tests with Fehling solution (hot' or cold), alcoholic silvernitrate, ferric chloride and 2,4-dinitrophenylhydrazine. Concentrated sulfuric acid is not colorized where .it is kept with the product at room temperature for 24 hours.'Bio.- logical activity of the product shows no significant change even after the product is heated in benzene at 80 ..;C. for 30 minutes.

Infrared. absorption at 3480 cm. confirms the existence of apyrrole nucleus in the product. 'Ihisis also con firmed by purple coloration of this product in Ehirlichs reaction and by nuclear magnetic resonance spectrum. Furthen'irifrared absorption appearing at 1530 and 1375 cm'r shows the existence of 'nitro group. Ultraviolet absorptionofthe compound is A 252 my. (e 7500) in ethanol. (See'FIGS. 1, 2 and 3'.) k 1' The decomposition tests of this compoundare as folows:

this as an upper layer -agar plates for bioassay are pre- "pared in the; mannefknown perse. Incubation for bioassay is carried out at 28-30 C. for 40'hours, and then,

antibiotic potency is assayed. Contaminationwith infec- --'tious bacteria which occasionally occurs duringfltheratlier long period of incubation can be preventedby'jdiluting a' test liquid with a'phosphoric acid-bulfered solution (pH 6.0), containing 0.1% chlorarnphenico'l, where acup lmethod is.employedorbynsing 'aidry pulp disc previously inipregnated" a methanolic solution-containing" 0.1% chloramphenicol where a pulp method ernployedlj Pyrrdlnit'rmQwhenintrape'ritoneally 'injectedft'o mice have an LD VaIue of 500 tr g/kg, No substantialchange insyst'erniccdndition and'body weight in'crease i'siobserved when pyrrolnitrin is administered to rats at the dosage of 30. mg./kg, .daily 7 over the. period of three months,

' In order toQsubstantiate the clinical utility of pyrrolnitrin, the following" clinical testsweref instituted. The pyrrolnitrin preparation used'was a solution containing 0.5% (w./v.) pyrrolnitrin in .7Q% by volume of 99% alcohol and 30% by'"volume of propyleneglyeol or" a solution con- 70% ;a1e ho1; 5n 2% ofdiethylj seb'acatef'fhis' alcoholic solution was applied ,to the alfected skin of a number of patients one or three-times daily. The result obtained are as follows:

NOTE.Very effective: Turn to negative for fungi under a microscope and complete disappearance of subjective and objective symptoms; Effective: Remarkable turn for the better in subjective and objective symptoms; Inefiective: No symptomatic changes.

As apparent from table, pyrrolnitrin was found to be 82.6 percent effective, and no substantial side effects were observed.

Pyrrolnitrin can be used in various medicinal forms such as spray solutions, tinctures, ointments, etc. It is most convenient to use it in the form of alcoholic aqueous solution, and in this form the substance is stable, even when stored at room temperature over one year. For topical application, pyrrolnitrin in concentration as 0.25- 3% will be sufiiciently effective. Furthermore, pyrrolnitrin may be used in preparations mixed with other medicaments.

Example 1 Pseudomonas pyrrocinia No. 2327 is inoculated to a nutrient agar slant culture medium, and incubation is carried out at 37 C. for 48 hours. Separately, 100 m1. of a culture medium which is prepared by dissolving 1% glucose, meat extract, 5% polypeptone and 2% sodium chloride in an aqueous solution containing 0.2 molar potassium dihydrogen phosphate and 0.2 molar sodium hydrogen phosphate is placed in a 500 ml. shake flask and then sterilized at 120 C. for minutes. The inoculum obtained by slant culture is inoculated in one platinum loop to the shake flask. Shaking culture of the inoculum is carried out at 30 C. for 24 hours. The culture broth thus prepared is inoculated in amount of 5% by volume to the shake flask containing the sterilized culture medium as above. Shake culture is effected at 30 C. for three days. The fermentation broth is divided to cells and a supernatant by centrifugation at 6500 r.p.m. The cell cake as formed is removed from the supernatant, and 60% aqueous acetone in amount of one-twentieth by volume of the broth is added thereto. Stirring is made at 50 C. for about 3 hours thereby to effect extraction of desired antibiotic substance with acetone. The acetone extract is recovered by filtration and then added with active carbon. Stirring is made for a short time to have impurities adsorbed with the active carbon. After filtration, the resulted extract is concentrated at about 40 C. under reduced pressure to make it one-fifth by volume. Using an equal volume of butyl acetate, the concentrated liquid is extracted three times. The butyl acetate layer is washed with aqueous alkali having pH 10.0 and then concentrated to have 100 mg. of an oily material. This is dissolved in methanol, and the resulted solution is added with five times by volume of cyclohexane. The resulting mixture is kept in an ice box. Desired product is obtained as yellow precipitate.

EXAMPLE 2 Into each of four glass jars (15 ml. volume), 8 ml. of a culture medium containing 0.5% meat extract, 0.5% peptone, 0.2% sodium chloride and 1% glucose and having a pH of 6.0 is charged, and sterilization is conducted at 120 C. for hours. The inoculum (Pseudomonas pyrrocinz'a No. 2327) obtained from the same culture medium as used in Example 1 by shake culture at 30 C. for 24 hours is inoculated in 5% by volume to each of the jars. Incubation is carried out at 28 C. with stirring at 330 r.p.m., while sterilized air is passed 10 into the jars. Vigorous foaming is prevented by adding sterilized soybean oil dropwise. After 48 hours, the fermentation broth is recovered, and cells are collected by means of a Sharples centrifugal machine. To the resulted cell cake, 70% aqueous acetone is added in amount of five times by volume based on the wet weight of the collected cells. After allowing the resulting mixture to stand for 24 hours at room temperature, the cells are filtered oif. The acetone extract is concentrated under reduced pressure to have a white turbid liquid concentrate which is in turn extracted with an equal volume of petroleum benzine. The resulting extract is washed Well with aqueous alkali, aqueous acid and water in sequence, and then concentrated. The resulted concentrated extract is dried and then passed through a column filled with alumina. Desired antibiotic substance adsorbed on alumina is washed by passing petroleum benzine and n-hexane through the column. Elution is made by using benzene. The effluents which are active against Tricophyton are collected and then concentrated under reduced pressure, thereby to obtain a syrup. This is dissolved in methanol. The methanolic solution is freed from impurities by filtration and then added with cyclohexane at the proportion of 3 parts per part of the methanolic solution. The mixture is kept in an ice box to have yellow precipitate. This is recrystallized from methanol-cyclohexane. Pure crystalline product obtained is 50 mg.

Example 3 Pseudomonas aeruginosa B16 strain is inoculated to a nutrient agar slant, and incubation is eiiected at 37 C. for 48 hours. ml. of a culture medium comprising 1% polypeptone, 1% meat extract, 0.5 sodium chloride, 2.65% potassium dihydrogen phosphate and 0.18% sodium dihydrogen phosphate (12 H O) is placed in a 500 ml. shaking flask and sterilized at C. for 15 minutes. The inoculum taken from the slant culture medium is inoculated in a platinum loop to the flask, and shaking culture is conducted at 37 C. for 24 hours. The culture broth thus obtained is inoculated to 10 l. of the culture medium of the same composition as above, in a 151. jar fermenter, said culture medium having been sterilized at 120 C. for 30 minutes. Cultivation is conducted at 30 C. for 3 days, with propellar agitation at 330 r.p.m., while sterilized air is passed thereinto at the rate of about 10 l. per minute.

The culture broth thus obtained is added with about 1% diatomaceous earth and the mixture is filtered to collect cells. The resulted cell cake is extracted with about 1 l. of 80% aqueous acetone under warming and then separated by filtration. The acetone extract obtained is concentrated under reduced pressure. The concentrate is then extracted three times with 200 ml. each of petroleum benzine. The extracts are combined and washed with an equal amount of water, decolorized with a small amount of active carbon and then concentrated to an oily material. This is passed into a column filled with alumina/ petroleum benzine. Desired antibiotic substance adsorbed on alumina is washed by passing petroleum benzine and n-hexane through the column, and then eluted with benzene. Among the eluted fractions, those which show high activity against Tricophyton are collected and then concentrated under reduced pressure to have a syrup. This is dissolved in methanol to remove undissolved matters. The solution obtained is added with cyclohexane at the proportion of 3 parts per part of the solution and the mixture is kept in an ice box. The yellow precipitate thus formed is collected and recrystallized from methanolcyclohexane. Pure crystalline product is obtained, M.P. C. Yield about 70 mg.

Example 4 Pseudomonas mephitica No. 29 strain is inoculated to a nutrient agar slant culture medium. Incubation is carried out at 30 C. for 48 hours. 100 ml. of a liquid culture medium comprising 1% polypeptone, 1% meat extract, 0.5 sodium chloride, 2.18% potassium dihydrogen phosphate and 1.43% disodium hydrogen phosphate (12H O) is placed in a 500 ml. shake flask and sterilized at 120 C. for 15 minutes. Into several shake flasks thus prepared, the inoculum obtained from the slant culture is inoculated in amount of one platinum loop. Shake culture is held at 30 C. for 24 hours. 'From the fermentation broth, cells are collected by a centrifugal separator operating at 6,500 r.p.m. The Wet cell cake is treated with 500 ml. of 100% acetone at 50 C. for 3 hours. After filtration, the extract is purified by treatment with active carbon. It is then concentrated to make its volume about one-sxith. The resulted concentrate is extracted three times with butyl acetate of the same quantity. The butyl acetate extract is washed with aqueous alkali (pH 10) and concenrated under reduced pressure to have an oily material, which is then worked up in the same manner as in Example 3. 30 mg. of pure crystalline product is obtained.

Example 5 Pseudomonas ovalis No. 103 is inoculated to a nutrient agar slant culture medium, and fected at 30 C. for 48 hours. 100 ml. of a liquid culture medium comprising 3% glycerine, 1% soybean meal, 0.05% magnesium sulfate, 0.3% sodium chloride, 2.18% potassium dihydrogen phosphate and 1.43 disodium hydrogen phosphate (12H O) is placed in each of several shake flasks (500 ml. volume), and sterilization is made at 120 C. 'for 15 minutes. To these flasks, the inoculum is inoculated from the above slant culture in amount of one platinum loop, and shake culture is held at 30 C. for 24 hours.

To each of the four glass jars used in Example 3, 8 l. of the same culture medium as specified above is charged, and sterilization is carried out at 120 C. for 30 minutes. To the culture medium thus prepared, the inoculum obtained as above is inoculated in seed volume of 2%, and cultivation is carried out at 30 C. for 24 hours with stirring, while sterilized air is passed therein at the rate of about 8 l./min. Under the sterilized condition, 301. of the culture broth recovered from the four jars is inoculated to 2500 l. of the same culture medium as above which is placed in a 4000 1. stainless steel fermenter, said culture medium having been sterilized at 120 C. for 40 minutes and then cooled down to 30 C. Cultivation is carriedout at 30 C. for 60 hours with propeller agitation at 180 r.p.m., while sterilized air is blown into the vessel in the equal amount to the culture medium at every minute. To the fermentation broth, about 2% of diatomaceous earth is added. Then the broth is filtered by means of -a filter press, and the cells collected are added with 300 l. of acetone. Extraction is effected at 40 C. for 3 hours with stirring. The procedures for extraction are repeated twice. The resulted acetone extract is concentrated at 40 incubation is ef- C. under reduced pressure to make its volume one-tenth. The concentrated liquid is extracted three times with 160 l. of petroleum benzine. The extract obtained is washed with an equal volume of water and decolorized with active carbon. .Then it is concentrated at 40 C. under reduced pressure to. have about 700 g. of an oily concentrate. This is extracted with 3 l. of ethyl acetate". The extract is concentrated to have an oily material which is then dissolved in 1500 ml. of n-hexane. The resulting solution is developed with n-hexane over 1600 g. of 100 mesh silicagel contained in a 10 cm. x cm. glass column. Elution of the effective substance is made by using n-hexanebenzene mixture. Trichophyton-active fractionsare collected. About 500 ml. of the collected fractions are concentrated under reduced pressure. The oily material obtained is added with n-hexane, and the mixture is allowed to standwith the result that about 11 g. of crude crystalline product is obtained. This is dissolved in about 200 ml. of benzene andthe resulted solution is subject to column chromatography using activated magnesium silicate. Elution is made by using benzene. Yellow fractions are collected and concentrated under reduced pressure thereby to yield 10 g. of pure crystalline product, M.P. 125 C.

Example 6 Pseudomonau shwylkilliensis -CB--416 inoculated to glycerine yeast extract agar slant, and incubation is carried out at 30 C. for 48 hours. ml. of a culture medium comprising 3% glycerine, 1% gluten meal, 1% dried yeast, 0.5% corn steep liquor (pH 7.0) and 0.025% FeSO -7H O is placed in each of two 500 ml. shake flasks and sterilized at C. for 15 minutes. The inoculum taken from the slant culture is inoculated in a pick of a platinum loop to the flasks, and shaking culture is carried out at 30 C. for 48 hours. The culture broth thus obtained is inoculated to 8 l. of the same culture medium as above which is placed in a 15 l. jar-fermenter, said culture medium having been sterilized at 120 C. for 30 minutes and then cooled. Cultivation is conducted at 30 C. for 96 hours with propeller agitation at 330 r.p.m., while sterilized air is passed thereinto. The fermentation broth thereby obtained is treated in the same manner as in Example 3 to obtain 138 mg. of pure crystalline product, M.P. C.

What we claim is:

1. 3-(2-nitro-3-chlorophenyl)-4-ch1oro-pyrrole.

References Cited JEROME D. GOLDBERG, Primary Examiner v US. Cl. X.R.

PO-Hm i UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,699,121 P Dted October 17 1972 I Inventor) Kei Arima, et al.

It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown,below:

r- Column 1, line 9, after "16,338" insert --and application 7 Japan, June 12, 1964, 39/33,177--.

Signed and sealed this 2 +th dsy of April 1973.

(SEAL) I fittest: I

EDWARD-M. FLETCHER, JR." ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents 

